Project Summary Opioids are used in treating chronic pain but have adverse effects including problematic issues of tolerance, dependence, and opioid-induced hyperalgesia. The best-established mechanism of opioid dependence is the up-regulation of adenylate cyclase (AC)/cAMP pathway. Recent studies revealed that hydrogen sulfide (H2S) can effectively attenuate the development of opioid dependence via down-regulation of the AC/cAMP pathway. However, H2S's exact mechanisms of action are still unclear, and the actual H2S concentration and flux which exhibit the optimal inhibition of opioid dependence have yet to be determined. This lack of knowledge presents a major burden in the development of H2S based therapy. In this regard, research tools for convenient and accurate detection of H2S are critically needed. In this project we plan to develop highly sensitive fluorescent sensors for the detection of H2S in living systems. We will also use these sensors to explore optimal H2S flux in a well-established cell model of opioid dependence. This study will not only clarify the role H2S plays in suppressing opioid dependence, but also establish useful protocols for the evaluation of H2S donors for the treatment. This study should guide future development of H2S-based therapy for opioid dependence.